Method for separating leukemia stem cells from leukemia cell strain

A technology of leukemia stem cells and leukemia cells, applied in the field of leukemia stem cells, can solve the problems of lack of LSCs, survival pathways, apoptosis pathways and self-renewal, etc.

Inactive Publication Date: 2011-10-19
GUANGDONG GENERAL HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, although the concept of leukemia stem cells has been proposed for many years, more and more attention has been paid to leukemia stem cells only in recent years, but the various properties of LSCs, such as survival pathways, apoptosis pathways, and self-renewal, are poorly understood.
The greatest limitation to the study of LSCs is the lack of sufficient LSCs

Method used

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  • Method for separating leukemia stem cells from leukemia cell strain
  • Method for separating leukemia stem cells from leukemia cell strain
  • Method for separating leukemia stem cells from leukemia cell strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1 Isolation of leukemia stem cells from leukemia cell lines

[0021] 1. Experimental materials

[0022] 1. Primary Solutions and Reagents

[0023] PRMI1640 liquid medium (Gibco, USA); fetal bovine serum (Sijiqing, Hangzhou); tissue grade DMSO, propidium iodide (PI) (Sigma Chemical Co, St. Louis, MO), CD38-FITC, anti-FITC Microbeads, BSA, EDTA, LD column, MACS separator (Miltenyi Biotec Company, Germany), CD34-APC, CD38-FITC, CD45-perCP, CD11b, CD123-PE ((Pharmingen, USA), Difco Bacto soft Agar (Allexis, USA), human-specific CD45 and CD34 antibodies (DAKO).

[0024] 2. Preparation of main reagent solutions

[0025] ① Cell cryopreservation medium: add 1 part of tissue-grade DMSO to 9 parts of fetal bovine serum to prepare 10% DMSO cell cryopreservation medium.

[0026] ②Phosphate-buffered saline (PBS): 8g Nacl+0.2g ​​KCl+ 2.8g NaCl 2 HPO 4 12H 2 O+0.2g ​​KH 2 PO 4 , with ultrapure water to a total volume of 1000ml, adjusted to a pH value of 7.4, aliquoted...

Embodiment 2

[0165] Sorted leukemia stem cells are resistant to conventional chemotherapy drugs and NK cell killing

[0166] 1. Experimental materials

[0167] PRMI 1640 liquid medium (Gibco, USA); fetal bovine serum (Sijiqing, Hangzhou); daunorubicin (Daunorubicin, DNR, Shenzhen Wanle Pharmaceutical Factory)) and mitoxantrone (Mitoxantrone, Mito, Zhejiang Ruixin Pharmaceutical Co., Ltd.), tissue grade DMSO, thiazolyl blue (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium, MTT) (Sigma Chemical Co, St. Louis, MO), LDH killing Activity detection kit (Promega). CD38-FITC, anti-FITC Microbeads, anti-CD56 immunomagnetic beads, BSA, EDTA, LD column, MACS separator (Miltenyi Biotec Company, Germany).

[0168] 2. Experimental method

[0169] 1. Isolation of Peripheral Blood Mononuclear Cells

[0170] Separation by conventional density gradient centrifugation: aseptically extract peripheral venous blood, anticoagulate with heparin (20U / ml blood), dilute with PBS at a volume of 3:1, carefull...

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Abstract

The invention provides a method for separating leukemia stem cells from a leukemia cell strain, which comprises the following steps: firstly, subculturing human acute myelogenous leukemia cell strain KD1a cells; and secondly, separating CD34+CD38-cells by an immunomagnetic bead separation method to obtain the leukemia stem cells, wherein the leukemia stem cells can resist conventional chemotherapy and NK immunization therapy. The separation method disclosed by the invention is simple to operate, can separate a large quantity of high-purity leukemia stem cells from KG1a cells, and has an important significance for the research of the leukemia stem cells.

Description

technical field [0001] The invention relates to a method for sorting leukemia stem cells, in particular to a method for sorting leukemia stem cells resistant to conventional chemotherapy and NK immunotherapy from leukemia cell lines. Background technique [0002] Acute myeloid leukemia (AML) is an acquired malignant clonal disease of hematopoietic stem cells with various genetic abnormalities. In recent years, the treatment of AML has undergone many changes. The application of CD33 monoclonal antibody, the emergence of new chemotherapy drugs and chemotherapy regimens have significantly increased the remission rate and re-remission rate after relapse. Allogeneic hematopoietic stem cell transplantation is still the only curative method for AML. However, due to the influence of donor sources, patient age, and other factors, most AML patients cannot receive allogeneic hematopoietic stem cell transplantation, and there are still risk factors such as graft-versus-host disease, gr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/095
Inventor 佘妙容陈锡林牛新清李劲高郭坤元杜欣周茂华林伟陆泽生
Owner GUANGDONG GENERAL HOSPITAL
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